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1.
Environ Pollut ; 302: 118961, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35183667

RESUMO

The intestinal microbiota has a key role in human health via the interaction with the somatic and immune cells in the digestive tract environment. Food, through matrix effect, nutrient and non-nutrient molecules, is a key regulator of microbiota diversity. As a food contaminant, the pesticide chlorpyrifos (CPF) has an effect on the composition of the intestinal microbiota and induces perturbation of microbiota. Prebiotics (and notably inulin) are known for their ability to promote an equilibrium of the microbiota that favours saccharolytic bacteria. The SHIME® dynamic in vitro model of the human intestine was exposed to CPF and inulin concomitantly for 30 days, in order to assess variations in both the bacterial populations and their metabolites. Various analyses of the microbiota (notably temporal temperature gradient gel electrophoresis) revealed a protective effect of the prebiotic through inhibition of the enterobacterial (E. coli) population. Bifidobacteria were only temporarily inhibited at D15 and recovered at D30. Although other potentially beneficial populations (lactobacilli) were not greatly modified, their activity and that of the saccharolytic bacteria in general were highlighted by an increase in levels of short-chain fatty acids and more specifically butyrate. Given the known role of host-microbiota communication, CPF's impact on the body's homeostasis remains to be determined.


Assuntos
Clorpirifos , Microbiota , Clorpirifos/toxicidade , Escherichia coli/metabolismo , Humanos , Inulina/metabolismo , Inulina/farmacologia , Prebióticos/análise
2.
PLoS Pathog ; 18(6): e1010584, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35696408

RESUMO

Escherichia coli F18 is a common conditional pathogen that is associated with a variety of infections in humans and animals. LncRNAs have emerged as critical players in pathogen infection, but their role in the resistance of the host to bacterial diarrhea remains unknown. Here, we used piglets as animal model and identified an antisense lncRNA termed FUT3-AS1 as a host regulator related to E. coli F18 infection by RNA sequencing. Downregulation of FUT3-AS1 expression contributed to the enhancement of E. coli F18 resistance in IPEC-J2 cells. FUT3-AS1 knockdown reduced FUT3 expression via decreasing the H4K16ac level of FUT3 promoter. Besides, the FUT3-AS1/miR-212 axis could act as a competing endogenous RNA to regulate FUT3 expression. Functional analysis demonstrated that target FUT3 plays a vital role in the resistance of IPEC-J2 cells to E. coli F18 invasion. A Fut3-knockout mice model was established and Fut3-knockout mice obviously improved the ability of resistance to bacterial diarrhea. Interestingly, FUT3 could enhance E. coli F18 susceptibility by activating glycosphingolipid biosynthesis and toll-like receptor signaling which are related to receptor formation and immune response, respectively. In summary, we have identified a novel biomarker FUT3-AS1 that modulates E. coli F18 susceptibility via histone H4 modifications or miR-212/FUT3 axis, which will provide theoretical guidance to develop novel strategies for combating bacterial diarrhea in piglets.


Assuntos
Infecções por Escherichia coli , MicroRNAs , RNA Longo não Codificante , Doenças dos Suínos , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Diarreia/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Regulação Neoplásica da Expressão Gênica , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Suínos , Doenças dos Suínos/genética
3.
Int J Biol Macromol ; 213: 555-564, 2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35644318

RESUMO

Fungal immunomodulatory proteins (FIPs) have been investigated for their use as potential natural derived anti-tumor molecules. However, the stability of FIPs is critical for their preparation and storage. In this study, the correlation between thermal stability and protein structural features of rFIP-nha, with significant anti-tumor activity, has been evaluated. For comprehensive analysis, FIP-nha and its homologues FIP-gmi, FIP-fve, and LZ-8 were all recombinantly expressed in E. coli. In solution, rFIP-nha and rFIP-gmi formed tetramers; rFIP-fve and rLZ-8 appeared as dimers. Their melting temperatures were 85.1 °C, 77.8 °C, 66.5 °C, and 64.4 °C, respectively. Accordingly, their cytotoxicity was also temperature dependent. To investigate the underlying mechanism of their thermostability, we solved the crystal structure of FIP-nha. Detailed structure analysis, molecular dynamic simulation and mutagenesis studies indicated that a higher thermostability was correlated to higher oligomerization states, larger interface area, and more interactions. The structure property studies indicate that Y12, D61 and Y108 were critical for oligomerization and high thermostability of rFIP-nha, but the dimeric and tetrameric states of rFIP-nha exert similar cytotoxicity on A549 cells. Taken together, these findings reveal that thermostability of FIPs was dependent on their oligomerization state, and correlated with their cytotoxicity.


Assuntos
Escherichia coli , Fusarium , Células A549 , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Fusarium/metabolismo , Humanos
4.
Antimicrob Agents Chemother ; 66(6): e0240221, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35647648

RESUMO

KPC-53 enzyme is a natural KPC variant which showed a duplication of L167E168 residues in the Ω-loop structure. The blaKPC-53 gene was cloned both into pBC-SK and pET-24a vectors, and the recombinant plasmids were transferred by transformation in Escherichia coli competent cells to evaluate the antimicrobial susceptibility and to produce the enzyme. Compared to KPC-3, the KPC-53 was less stable and showed a dramatic reduction of kcat and kcat/Km versus several ß-lactams, in particular carbapenems. Indeed, a 2,000-fold reduction was observed in the kcat values of KPC-53 for imipenem and meropenem. Concerning inhibitors, KPC-53 was susceptible to tazobactam and clavulanic acid but maintained resistance to avibactam. The molecular modeling indicates that the L167E168 duplication in KPC-53 modifies the interactions between residues involved in the catalytic pocket, changing the flexibility of the Ω-loop, which is directly coupled with the catalytic properties of the KPC enzymes.


Assuntos
Aminoácidos , beta-Lactamases , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/metabolismo , Combinação de Medicamentos , Escherichia coli/metabolismo , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo
5.
J Agric Food Chem ; 70(24): 7460-7470, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35671140

RESUMO

Walnut (Juglans regia L.) cake meal constitutes a significant amount of solid byproduct from the production of walnut oil, comprising more than 40% protein. However, it is usually not well utilized. Therefore, an antibacterial peptide was obtained by hydrolyzing walnut oil residue protein with pepsin based on the diameter parameters of the antibacterial zone in this research. The purified antibacterial peptide WRPH-II-6 was obtained by two-part purification (ultrafiltration and reversed-phase liquid chromatography) and possessed higher antibacterial activity against Escherichia coli (MIC = 1.33 mg/mL), Staphylococcus aureus (MIC = 0.33 mg/mL), and Bacillus subtilis (MIC = 0.66 mg/mL). The amino acid sequence of WRPH-II-6 was identified as TGSAVPSPRASATATMEMAAAMGLMPGSPSSVSAVMSPF, where the presence of a large proportion of hydrophobic amino acid residues, such as alanine, proline, and methionine, explained the marked antibacterial activity of WRPH-II-6. The harsh sensitivity experiment demonstrated that WRPH-II-6 retains the stability of antibacterial activity when exposed to broad-spectrum pH values, variable temperatures, and long-lasting UV irradiation. The antibacterial mechanism of the WRPH-II-6 peptide against S. aureus and B. subtilis involves nonmembrane disruption: the contact of anions and cations causes the folding and collapse of the bacterial cell membrane to achieve the inhibitory effect. The antibacterial mechanism against E. coli is membrane disruption, which markedly disrupts the bacterial cell membrane to achieve the bactericidal effect. Significantly, the walnut residual protein hydrolysate is a potent preservative and antibacterial agent.


Assuntos
Juglans , Staphylococcus aureus , Antibacterianos/química , Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Resíduos Industriais , Juglans/química , Testes de Sensibilidade Microbiana , Peptídeos/química
6.
Methods Mol Biol ; 2487: 1-13, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687226

RESUMO

Many biosynthetic transformations have strict spatial and temporal requirements that necessitate the physical association of multiple enzymes for proper function. Here, we describe protocols for obtaining large multienzyme assemblies (>500 kDa) by recombinant expression in Escherichia coli. We focus on assemblies from stand-alone enzymes joined by intermolecular forces rather than multiple catalytic domains from a single polypeptide chain. Details are given for strategies to optimize protein expression and to design a multi-affinity tag purification scheme for large multienzyme assemblies. These insights are drawn from our study of bacterial hydrocarbon biosynthesis.


Assuntos
Escherichia coli , Domínio Catalítico , Cromatografia de Afinidade/métodos , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Methods Mol Biol ; 2487: 197-204, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687238

RESUMO

Multienzyme complexes naturally exist in cells to catalyze cascade reactions in metabolic pathways. By clustering the enzymes in close proximity, these nanomachineries achieve effective conversion of metabolites. Bioengineers are working on the development of synthetic versions of multienzyme complexes in cells to synergize heterologous biosynthesis. Assembling enzymes on protein scaffolds through protein-protein interactions is a viable and facile way to form synthetic multienzyme complexes. Here, we describe the general methods to construct self-assembled multienzyme nanostructures in Escherichia coli for biosynthesis of valuable chemicals.


Assuntos
Nanoestruturas , Biocatálise , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Complexos Multienzimáticos/química , Nanoestruturas/química , Proteínas/química
8.
Methods Mol Biol ; 2487: 297-315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35687243

RESUMO

The utility of ATP-dependent multi-enzymatic reactions is limited by their requirement for stoichiometric amounts of this expensive cofactor or additional purified enzymes for its recycling. Here we describe a simple method for the production of recombinant cell-free extracts (or lysates) of E. coli that support ATP-dependent biotransformations. The inexpensive preparation described is obtained with modest processing from a single recombinant bacterial culture of E. coli. In addition to recombinantly overexpressed enzymes that catalyze the primary ATP-dependent reactions of interest, endogenous kinases that are naturally present in the extract catalyze recycling of the requisite ATP. This means that only catalytic amounts of cofactor are necessary to drive the biotransformation, and without the requirement for additional purified enzymes. This approach has been applied successfully to an array of in vitro enzymatic cascades with multiple ATP-dependent steps.


Assuntos
Trifosfato de Adenosina , Escherichia coli , Trifosfato de Adenosina/metabolismo , Biocatálise , Catálise , Escherichia coli/metabolismo
9.
Molecules ; 27(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35684325

RESUMO

Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal-protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.


Assuntos
Proteínas de Escherichia coli , Helicobacter pylori , Sítios de Ligação , Cobre/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Helicobacter pylori/metabolismo , Metionina/metabolismo
10.
Methods Mol Biol ; 2528: 31-37, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35704183

RESUMO

Early evidence for R-loop formation in vivo came from the study of Escherichia coli topA (topoisomerase I; topo I) null mutants. Assays with plasmids to detect RNase HI-sensitive hypernegative supercoiling or R-looped DNA were used in vitro and in vivo to demonstrate R-loop formation. In addition, these R-loop-dependent topological modifications of plasmid DNA were shown to correlate with severe growth and gene expression inhibition in topA null mutants that could be corrected by RNase HI overproduction. However, direct evidence for R-loop formation on chromosomal DNA from E. coli cells was only obtained recently by using the S9.6 antibody to detect RNA-DNA hybrids in dot-blot experiments. Here, we present a protocol for such experiments with a special emphasis on the procedure used for bacterial genomic DNA extraction and preparation including treatment with appropriate ribonucleases to eliminate RNA-RNA hybrids (that are also recognized by S9.6) as well as single-stranded RNA (ssRNA), in order to obtain a signal that is specific to stable RNA-DNA hybrids generated. Furthermore, we recommend that the results of such experiments be correlated with RNase HI-sensitive phenotypes.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética , Estruturas R-Loop , RNA , Ribonuclease H/genética , Ribonuclease H/metabolismo
11.
Microb Cell Fact ; 21(1): 97, 2022 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-35643494

RESUMO

BACKGROUND: Carbamate pesticides have been widely used in agricultural and forestry pest control. The large-scale use of carbamates has caused severe toxicity in various systems because of their toxic environmental residues. Carbaryl is a representative carbamate pesticide and hydrolase/carboxylesterase is the initial and critical enzyme for its degradation. Whole-cell biocatalysts have become a powerful tool for environmental bioremediation. Here, a whole cell biocatalyst was constructed by displaying a novel carboxylesterase/hydrolase on the surface of Escherichia coli cells for carbaryl bioremediation. RESULTS: The carCby gene, encoding a protein with carbaryl hydrolysis activity was cloned and characterized. Subsequently, CarCby was displayed on the outer membrane of E. coli BL21(DE3) cells using the N-terminus of ice nucleation protein as an anchor. The surface localization of CarCby was confirmed by SDS-PAGE and fluorescence microscopy. The optimal temperature and pH of the engineered E. coli cells were 30 °C and 7.5, respectively, using pNPC4 as a substrate. The whole cell biocatalyst exhibited better stability and maintained approximately 8-fold higher specific enzymatic activity than purified CarCby when incubated at 30 °C for 120 h. In addition, ~ 100% and 50% of the original activity was retained when incubated with the whole cell biocatalyst at 4 ℃ and 30 °C for 35 days, respectively. However, the purified CarCby lost almost 100% of its activity when incubated at 30 °C for 134 h or 37 °C for 96 h, respectively. Finally, approximately 30 mg/L of carbaryl was hydrolyzed by 200 U of the engineered E. coli cells in 12 h. CONCLUSIONS: Here, a carbaryl hydrolase-containing surface-displayed system was first constructed, and the whole cell biocatalyst displayed better stability and maintained its catalytic activity. This surface-displayed strategy provides a new solution for the cost-efficient bioremediation of carbaryl and could also have the potential to be used to treat other carbamates in environmental bioremediation.


Assuntos
Escherichia coli , Praguicidas , Biodegradação Ambiental , Carbaril/metabolismo , Carboxilesterase/genética , Carboxilesterase/metabolismo , Escherichia coli/metabolismo , Praguicidas/metabolismo
12.
Microb Cell Fact ; 21(1): 104, 2022 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-35643507

RESUMO

Listeria monocytogenes is a food-borne pathogen. Pediocin is a group IIα bacteriocin with anti-listeria activity that is naturally produced by Pediococcus acidilactic and Lactobacillus plantarum. The pedA/papA gene encodes pediocin/plantaricin. In native hosts, the expression and secretion of active PedA/PapA protein rely on the accessory protein PedC/PapC and ABC transporter PedD/PapD on the same operon. The excretion machines were also necessary for pediocin protein expression in heterologous hosts of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vectors carrying the codon sequence of the mature PapA peptide were constructed, one with and one without a His tag. Both fragments were inserted into the plasmid pHT43 and transformed into Bacillus subtilis WB800N. The strains were induced with IPTG to secrete the fused proteins PA1 and PA2. Supernatants from both recombinant strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole dispense with removal of the leading peptide. This is the first report of active pediocin/PapA expression without the assistance of PedCD/PapCD in heterogeneous hosts. In addition, the PA1 protein can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography.


Assuntos
Bacillus subtilis , Bacteriocinas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacteriocinas/genética , Bacteriocinas/farmacologia , Escherichia coli/metabolismo , Pediocinas/metabolismo , Pediococcus/genética , Pediococcus/metabolismo
13.
Methods Enzymol ; 669: 3-27, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35644177

RESUMO

Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases catalyze key steps in the biosynthesis of numerous biomolecules, including protein cofactors, antibiotics, herbicides, and other natural products, but have remained a relatively understudied subclass of radical SAM enzymes due to their inherent insolubility upon overproduction in Escherichia coli. These enzymes contain two cofactors: a [4Fe-4S] cluster that is ligated by three cysteine residues, and a cobalamin cofactor typically bound by residues in the N-terminal portion of the enzyme. Recent advances in the expression and purification of these enzymes in their active states and with both cofactors present has allowed for more detailed biochemical studies as well as structure determination by X-ray crystallography. Herein, we use KsTsrM and TokK to highlight methods for the structural characterization of cobalamin-dependent radical SAM (RS) enzymes and describe recent advances in in the overproduction and purification of these enzymes.


Assuntos
S-Adenosilmetionina , Vitamina B 12 , Cristalografia por Raios X , Escherichia coli/metabolismo , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo
14.
Front Cell Infect Microbiol ; 12: 885191, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35706909

RESUMO

Typical enteroaggregative Escherichia coli (tEAEC) is a diarrheagenic E. coli pathotype associated with pediatric and traveler's diarrhea. Even without diarrhea, EAEC infections in children also lead to increased gut inflammation and growth shortfalls. EAEC strain's defining phenotype is the aggregative adherence pattern on epithelial cells attributable to the aggregative adherence fimbriae (AAF). EAEC only causes diarrhea in humans; therefore, not much is known of the exact intestinal region of infection and damage or its interactions with intestinal enterocytes in vivo and in situ. This study aimed to develop a new tEAEC mouse model of infection, characterize the microbiota of infected mice, and evaluate in situ the expression of host adherence and surface molecules triggering EAEC infection and the role of the EAEC AAF-II in adherence. Six-week-old C57BL/6 mice, without previous antibiotic treatment, were orally challenged with EAEC 042 strain or EAEC 042 AAF-II mutant (ΔAAF/II) strain, or DAEC-MXR strain (diffusely adherent E. coli clinical isolate), and with saline solution (control group). Paraffin sections of the colon and ileum were stained with H&E and periodic acid-Schiff. ZO-1, ß-catenin, MUC1, and bacteria were analyzed by immunofluorescence. EAEC-infected mice, in comparison with DAEC-MXR-infected and control mice, significantly lost weight during the first 3 days. After 7 days post-infection, mucus production was increased in the colon and ileum, ZO-1 localization remained unaltered, and morphological alterations were more pronounced in the ileum since increased expression and apical localization of ß-catenin in ileal enterocytes were observed. EAEC-infected mice developed dysbiosis 21 days post-infection. At 4 days post-infection, EAEC strain 042 formed a biofilm on ileal villi and increased the expression and apical localization of ß-catenin in ileal enterocytes; these effects were not seen in animals infected with the 042 ΔAAF/II strain. At 3 days post-infection, MUC1 expression on ileal enterocytes was mainly detectable among infected mice and colocalized with 042 strains on the enterocyte surface. We developed a novel mouse model of EAEC infection, which mimics human infection, not an illness, revealing that EAEC 042 exerts its pathogenic effects in the mouse ileum and causes dysbiosis. This model is a unique tool to unveil early molecular mechanisms of EAEC infection in vivo and in situ.


Assuntos
Infecções por Escherichia coli , Íleo , Microbiota , Mucina-1 , beta Catenina , Adesinas de Escherichia coli/genética , Animais , Aderência Bacteriana/genética , Diarreia/microbiologia , Modelos Animais de Doenças , Disbiose , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mucina-1/genética , Muco/metabolismo , Viagem , beta Catenina/genética
15.
PLoS One ; 17(6): e0270097, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35714106

RESUMO

Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) proteins, which function to protect other "client" proteins in trans from various stresses in vitro and in vivo. Here, we show that fusion of Hero proteins in cis can enhance the molecular property of recombinant proteins. Fusion with Hero11 improved the otherwise challenging production of TAR DNA-binding protein of 43 kDa (TDP-43) in Escherichia coli. Moreover, fusing with Hero9 strongly protected the activity of firefly luciferase bearing destabilizing mutations against heat and other stress conditions. These data suggest that Hero proteins have the potential to be used as versatile stabilization tags for recombinant protein production.


Assuntos
Escherichia coli , Temperatura Alta , Biotecnologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade
16.
Int J Biol Macromol ; 213: 1007-1017, 2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35690161

RESUMO

The COVID-19 pandemic has highlighted the need for new vaccine platforms to rapidly develop solutions against emerging pathogens. In particular, some plant viruses offer several advantages for developing subunit vaccines, such as high expression rates in E. coli, high immunogenicity and safety, and absence of pre-immunity that could interfere with the vaccine's efficacy. Cowpea chlorotic mottle virus (CCMV) is a model system that has been extensively characterized, with key advantages for its use as an epitope carrier. In the present study, three relevant epitopes from the SARS-CoV-2 Spike protein were genetically inserted into the CCMV CP and expressed in E. coli cultures, resulting in the CCMV1, CCMV2, and CCMV3 chimeras. The recombinant CP mutants were purified from the formed inclusion bodies and refolded, and their immunogenicity as a subunit vaccine was assessed in BALB/c mice. The three mutants are immunogenic as they induce high IgG antibody titers that recognize the recombinant full-length S protein. This study supports the application of CCMV CP as an attractive carrier for the clinical evaluation of vaccine candidates against SARS-CoV-2. Furthermore, it suggests that VLPs assembled from these chimeric proteins could result in antigens with better immunogenicity.


Assuntos
Bromovirus , COVID-19 , Animais , Bromovirus/genética , Bromovirus/metabolismo , COVID-19/prevenção & controle , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Quimera/metabolismo , Epitopos , Escherichia coli/metabolismo , Humanos , Camundongos , Pandemias , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus , Vacinas de Subunidades
17.
Methods Mol Biol ; 2461: 137-147, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35727448

RESUMO

Development of strong promoters is of growing interest in the field of biotechnology and synthetic biology. Here we present a protocol for the construction of strong prokaryotic promoters that can be recognized by designated multiple sigma factors by interlocking their cognate binding motifs on DNA strands. Strong and stress responsive promoters for Escherichia coli and Bacillus subtilis have been created following the presented protocol. Customized promoters could be easily developed for fine-tuning gene expression or overproducing enzymes with prokaryotic cell factories.


Assuntos
Bacillus subtilis , Fator sigma , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismo
18.
Methods Mol Biol ; 2498: 283-292, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35727550

RESUMO

The production of recombinant proteins in bacteria made possible to obtain large quantities of proteins essential for basic and applied research. Escherichia coli remains one of the organisms of choice for recombinant proteins because of its ability to grow at high density and availability of a vast catalog of cloning vectors and mutant host strains. Here, we describe the protocols for the expression of cold-adapted (hemo)globins in Escherichia coli.


Assuntos
Escherichia coli , Globinas , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Globinas/metabolismo , Proteínas Recombinantes/metabolismo
19.
Microb Cell Fact ; 21(1): 125, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729580

RESUMO

BACKGROUND: Generally, bacteria have a circular genome with a single replication origin for each replicon, whereas archaea and eukaryotes can have multiple replication origins in a single chromosome. In Escherichia coli, bidirectional DNA replication is initiated at the origin of replication (oriC) and arrested by the 10 termination sites (terA-J). RESULTS: We constructed E. coli derivatives with additional or ectopic replication origins, which demonstrate the relationship between DNA replication and cell physiology. The cultures of E. coli derivatives with multiple replication origins contained an increased fraction of replicating chromosomes and the cells varied in size. Without the original oriC, E. coli derivatives with double ectopic replication origins manifested impaired growth irrespective of growth conditions and enhanced cell size, and exhibited excessive and asynchronous replication initiation. The generation time of an E. coli strain with three replication origins decreased in a minimal medium supplemented with glucose as the sole carbon source. As well as cell growth, the introduction of additional replication origins promoted increased biomass production. CONCLUSIONS: Balanced cell growth and physiological stability of E. coli under rapid growth condition are affected by changes in the position and number of replication origins. Additionally, we show that, for the first time to our knowledge, the introduction of replication initiation sites to the chromosome promotes cell growth and increases protein production.


Assuntos
Cromossomos Bacterianos , Escherichia coli , Biomassa , Cromossomos Bacterianos/genética , Replicação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Origem de Replicação
20.
Sci Rep ; 12(1): 10336, 2022 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-35725867

RESUMO

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.


Assuntos
Escherichia coli , Pseudomonas stutzeri , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfatos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Pseudomonas stutzeri/metabolismo
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